Unraveling of the whole human genome has shifted the focus of interest of scientists and researchers on the analysis of proteins; i.e., gene products. It may not be overstating to say that substantial protein analysis can be made possible only when a molecule that exhibits affinity (binding property) for each protein of interest has been successfully obtained. Cells, however, each contain quite many different types of proteins, and the amino acid sequence and structure of many of which are still unknown.
The most common technique for obtaining a molecule that exhibits affinity for a specific protein is to prepare an affinity antibody by utilizing the immune system of animal. However, this technique uses animals and thus, requires a large quantity of proteins, a large number of steps and large cost. Additionally, no affinity antibody can be obtained for specific substances with this technique. A technique called the aptamer method (also referred to as the SELEX method) that does not rely on any living organism has been proposed to avoid this problem. However, while a molecule obtained by this technique strongly interacts with a specific protein, this technique is not applicable to all the proteins.
In view of such circumstances, the present inventors proposed a modified aptamer method that is established by improving the aptamer method so as to use nucleic acids (see International Publication No. WO 2003/078623). However, since the modified aptamer method uses a number of different modified nucleic acids, and thus, it has been difficult to find appropriate PCR conditions. Additionally, the above method poses a problem that a functional molecule that tends to be strongly bound to a target substance is hard to be amplified by PCR. In order to solve the above existing problems, the present inventors have previously proposed a method for synthesizing an amidite (raw material) of a functional molecule in which the functional groups participating in binding to the target substance correspond one-to-one to the sequences of the dimers, and the functional groups are removed after binding to proteins and then, the resultant product can be amplified by PCR.
Meanwhile, a solid-phase synthesis of nucleic acids has been performed for 20 years or longer, and an automated synthesizer employing it was also sold at that time. The solid-phase synthesis of nucleic acids is performed by, for example, condensating nucleoside compounds (amidites) with nucleosides bound to a solid-phase support (e.g., CPG). During this condensation reaction, it is necessary that only the phosphoric acid moiety of each amidite is condensated with only the hydroxyl group of another amidite so that the other reactive groups do not participate in the condensation reaction. Thus, protecting groups are introduced to the reactive groups (e.g., exocyclic amino groups of nucleic acid bases of amidites used and a phosphoric acid moiety which is not made to participate in the condensation reaction) so that they do not participate in the condensation reaction, and the protecting groups are removed (deprotected) after completion of the whole condensation reaction. Conventionally, a benzoyl group, an isobutyryl group, other groups have been used as a protecting group which is introduced to the exocyclic amino group of a nucleic acid base, and these protecting groups are generally removed by treating the obtained nucleic acid with concentrated aqueous ammonia at 55° C. for 8 hours to 15 hours.
However, in the production of nucleic acids having affinity (binding property) for proteins, under such conventional deprotection conditions, not only the protecting groups but also their modified moieties (substituents having binding property for proteins) are removed, resulting in that modified nucleic acids cannot be stably produced. Thus, in the production of such modified nucleic acids, in order to prevent the substituents having binding property for proteins from being removed together with the protecting groups, there is a need to use amidites having protecting groups which is capable of being removed under milder conditions.
For example, some conventional literatures report nucleic acid amidites having protecting groups which is capable of being removed by diazabicycloundecene (DBU) (i.e., a bulky base) (Acta. Chem., Scand., B37, 263 (1983) and J. Org. Chem., 54, 1,657 (1989)). But, these nucleic acid-synthesizing amidites are not stable in acetonitrile (i.e., an aprotic solvent) (Tetrahedron Letters 46, 6,729 (1990)) and are not suitable to practical use. Other literatures report nucleic acid-synthesizing amidites having protecting groups which is capable of being removed in pyridine using 0.5M DBU for 16 hours (Tetrahedron 48, 4,171 (1992) and Nucleosides & Nucleotides 13, 2,059 (1994)). But, the use of a high concentration of DBU and the deprotection for a long time problematically cause alkylation of the base of nucleic acid. Other literatures report nucleic acid-synthesizing amidites having protecting groups which is capable of being removed in methanol using K2CO3 (Tetrahedron Letters 46, 6,729 (1990) and Nucleic Acids Research 21, 3,493 (1993)). But, use of K2CO3 (a base) in methanol (a protic solvent) problematically causes decomposition of the esters, etc.
Therefore, demand has arisen for the developments of an excellent protecting group which is capable of being removed under mild conditions and with which a nucleic acid suitable for the analyses of target substances (e.g., proteins) can be consistently produced; a nucleic acid-synthesizing amidite having such protecting group; and a nucleic acid-synthesizing method using the nucleic acid-synthesizing amidite.